Our long-continued interest in the development of the small intestine is now focussed on the differentiation of the small intestine of the chick embryo in organ culture. Using a system in which intestinal explants survive for at least a week in a chemically defined medium without hormones, we have found that several aspects of differentiation are accelerated relative to their time of occurrence in the intact embryo. Thyroxine in physiological concentration causes a strong but unbalanced stimulation, resulting in over-production of microvilli that break down into vesicles that carry much of the alkaline phosphatase and disaccharidases produced into the medium. Proceeding from these findings we plan to continue studying the effects of inhibitors of RNA or protein synthesis, as well as effects of other hormones, in combination with thyroxine, on ultrastructural and enzymic aspects of differentiation. Techniques to be used include scanning and transmission electron microscopy, and quantitative and autoradiographic determination of incorporation of tritiated precursors into intact tissue and vesicles, in addition to light microscopy and routine measurement of enzyme activity. By such studies of the differentiating epithelial cell and the products it releases into the medium, under various regulatory regimens, we expect to penetrate further into differentiation, with particular reference to the synthesis of microvillus membrane in correlation with its constituent enzymes, and in relation to other components necessary for the formation of stable microvilli. We also hope to acquire more evidence about the nature of the inhibitory system that appears to restrain the pace of differentiation in the intact intestine.